Synthesis and Assembly of a Cholera Toxin B Subunit-Rotavirus VP7 Fusion Protein in Transgenic Potato

Abstract

A gene encoding VP7, the outer capsid protein of simian rotavirus SA11, was fused to the carboxyl terminus of the cholera toxin B subunit gene. A plant expression vector containing the fusion gene under control of the mannopine synthase P2 promoter was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB::VP7 fusion gene was detected in the genomic DNA of transformed potato leaf cells by polymerase chain reaction (PCR) amplification methods. Immunoblot analysis of transformed potato tuber tissue extracts showed that synthesis and assembly of the CTB::VP7 fusion protein into oligomers of pentameric size occurred in the transformed plant cells. The binding of CTB::VP7 fusion protein pentamers to sialo-sugar containing GM1 ganglioside receptors on the intestinal epithelial cell membrane was quantified by enzyme-linked immunosorbent assay (ELISA). The ELISA results showed that the CTB::VP7 fusion protein made up approx 0.01% of the total soluble tuber protein. Synthesis and assembly of CTB::VP7 monomers into biologically active pentamers in transformed potato tubers demonstrates the feasibility of using edible plants as a mucosal vaccine for the production and delivery system for rotavirus capsid protein antigens.

Expression and Characterization of Synthetic Heat-Labile Enterotoxin B Subunit and Hemagglutinin–Neuraminidase-Neutralizing Epitope Fusion Protein in Escherichia coli and Tobacco Chloroplasts

“…These compounds may bond or bridge to the recombinant LTB-HNE, and the results may appear as the oligomers mainly detected in tobacco leaf extracts. Similar results have also been observed in other studies in which several oligomers of LTB and CTB were expressed in tobacco plastids (Scott and Wilkinson 1999;Choi et al 2005). Further study is needed to clarify the answer to this question.…”

“…Transgenic plants have been studied as delivery systems for proteins of vaccines of pharmaceutical interest (Walmsley et al 2003;Choi et al 2005). Moreover, plants with plastid genetic engineering are known as a technology that offers the advantage of high protein expression levels.…”

“…In brief, wild-type LTB was cloned at the N-terminus of the NdeI site and at the Cterminus at the XhoI (pET21a-LTB) site. To subclone wildtype LTB into pET21a, the NdeI-XhoI-digested polymerase chain reaction (PCR) product regenerated by the pMYO46 plasmid (Choi et al 2005) was directly cloned into expression vector pET21a digested with NdeI and XhoI. The sequence of the forward primer is 5′-GGAATTCCA TATGAATAAA-GTAAAATGTTATGTTTTAT-3′ and that of the reverse primer is 5′-CCGCTCGAGGTT-TTCCA TACTGATTGCCGCA-3′ (restriction sites are underlined).…”

“…Recently, the successful use of plants as expression systems for a number of pathogen antigens has paved the way for newer methods of oral vaccine production (Walmsley et al 2003;Choi et al 2005;Kang et al 2006). In particular, plants with plastid genetic engineering offer several potentially attractive advantages, including high protein expression levels (De Cosa et al 2001;Tregoning et al 2003;Molina et al 2004), easy transgene stacking in operons , biosafety for maternal inheritance (Scott and Wilkinson 1999;Daniell 2002), and the absence of toxic effects of foreign proteins (Bogorad 2000;Daniell et al 2001).…”

Production of Human Rotavirus and Salmonella Antigens in Plants and Elicitation of fljB-Specific Humoral Responses in Mice

“…These higher yields compared to single antigens could be due to the fusion of the VP antigens with the fljB fragment. Previous efforts to produce rotavirus antigens in plants have been reported [33][34][35][36]. However, none of these studies showed the expression of VP4D or fljB or the fusion of rotavirus antigens with fljB.…”